Summary The structure of a virus particle must conform to the mechanism by which it enters and infects a cell. We seek to relate structures of virus particles and viral surface proteins to mechanisms of the steps in cell attachment, uptake and penetration that initiate a productive infection. The proposed experiments take advantage of recent advances in electron cryomicroscopy (cryo-EM) and live-cell imaging to define these mechanisms for both non- enveloped and enveloped viruses, taking rotaviruses and flaviviruses (West Nile and dengue viruses, in particular) as specific examples. Previous live-cell imaging studies defined the cellular entry pathway for rhesus rotavirus (RRV). We can now relate structures we have determined for distinct conformations of the spike-like viral protein 4 (VP4), which mediates cell entry, to stages of plasma-membrane remodeling in the entry pathway. We will extend on-going analyses by cryo-EM of VP4 conformations at the membrane interface, both on cells and in an in vitro-reconstituted system for early steps, to determine how RRV deforms and then disrupts a membrane bilayer. A mechanism for RRV membrane perforation, suggested by preliminary data, may help unify observations made with several other RNA viruses. Proposed experiments will connect high- resolution structures with direct analyses (by electron cryotomography and potentially by high-resolution template matching) of VP4 conformations during cell entry and with sequences of events in time (by lattice- light-sheet microscopy). For the flavivirus fusion protein (the E protein), completing a structure for the postfusion conformation of the intact protein will enable design of experiments to trap fusion intermediates and thus to define the fusion mechanism more completely than hitherto possible. The intended outcomes of the studies, for both rotaviruses and flaviviruses, are structurally and mechanistically accurate molecular movies of viral entry.